Understanding the diverse biochemistry of the chlorite dismutase family: from O2 to heme

了解亚氯酸盐歧化酶家族的多样化生物化学：从 O2 到血红素

• 批准号: 8964883
• 负责人: Jennifer L DuBois
• 金额: $ 30.44万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964883
• 关键词: Accounting; Address; Aerobic; Air; Ally; Anthrax disease; Antibiotic Resistance; Archaea; Bacteria; Basic Science; Binding; Biochemical; Biochemical Pathway; Biochemistry; Biology; Carbon; Carbon Dioxide; Catalysis; Cell Respiration; Cell physiology; Cells; Chemistry; Complex; Computing Methodologies; Data; Decarboxylation; Development; Disease; Electrons; Environment; Enzymes; Eukaryota; Eukaryotic Cell; Evolution; Family; Genes; Genetic; Goals; Gram-Positive Bacteria; Growth; Health; Heme; Hydrogen; Hydrogen Peroxide; Individual; Infection; Informatics; Isotopes; Kinetics; Life; Link; Listeriosis; Metabolism; Metals; Methods; Mission; Modeling; Molecular Chaperones; Movement; National Institute of General Medical Sciences; Nature; Organism; Pathway interactions; Perchlorates; Peroxides; Phase; Phenotype; Plague; Porphyrins; Positioning Attribute; Propionates; Protein Family; Proteins; Protons; Public Health; Reaction; Recombinants; Research; Respiration; Series; Side; Staphylococcus aureus; Structure; System; Testing; Time; Tuberculosis; Variant; Virulence; Work; abstracting; base; biodefense; chlorite; cofactor; coproporphyrinogen oxidase; enzyme substrate; ferrochelatase; ferryl iron; heme a; heme biosynthesis; innovation; insight; methicillin resistant Staphylococcus aureus; microbial; novel; pathogen; peroxidation; protonation; protoporphyrin IX; public health relevance; tool

 DESCRIPTION (provided by applicant): Heme is essential for aerobic life and cellular respiration. The pathway by which eukaryotic cells make heme has been known for some time. Prokaryotic heme biosynthesis, by contrast, has been harder to describe. Recently, a pathway for heme biosynthesis that fills all the remaining gaps has been proposed for Gram- positive bacteria. This is a group of organisms that includes numerous important pathogens that are threats to public health and biodefense, such as the causative agents of MRSA, TB, anthrax, and plague. The pathway differs from the canonical one in its final three steps, with the greatest departure at its terminus. The last step is a double oxidative decarboxylation catalyzed by enzymes known as HemQs: a novel subtype of chlorite dismutases (Clds). The latter are heme enzymes that detoxify the chlorite end product of perchlorate respiration, converting it to Cl- to O2. The initial phase of this research resulted in a rigorous description of the structure, mechanism, and biology of O2-generating Clds from both perchlorate respirers and non-respiring pathogens. Leveraging the tools, insights, and scientific team assembled via work on Clds, this proposal aims at providing a description of HemQ function at the level of the individual molecule and extending to the cellular context. As preliminary work, a hemQ strain of Staphylococcus aureus has been generated and shown to be a heme auxotroph and small colony variant (SCV): a phenotype associated with intracellular persistence and antibiotic resistance. In tandem, the HemQ enzyme from S. aureus has been shown to oxidatively decarboxylate two of the four propionate side chains of coproheme III, in a reaction that depends strictly H2O2. Focusing on the S. aureus system, Aim 1 is to understand how HemQ binds and activates coproheme toward oxidative decarboxylation, producing structural and energetic models of SaHemQ in complex with its substrate (coproheme III), intermediate (harderoheme) and product (heme b). Aim 2 is to test a mechanism for HemQ's reaction, in which coproheme is both substrate and cofactor in the peroxidation. Time-resolved and kinetic isotope methods will be used to examine a series of hypotheses invoking a ferric-hydroperoxy intermediate and intramolecular hydrogen atom transfer. Finally, aim 3 uses genetic, cell-based, and biochemical methods to understand HemQ's function in the context of the cell and evolution. We expect completion of the proposed work to define the ultimate step of a pathway that is absolutely fundamental to aerobic life, essential for robust pathogenic growth, and clinically connected to the development of persistence and antibiotic resistance.

 描述（由适用提供）：血红素对于有氧生活和细胞呼吸至关重要。真核细胞使血红素的途径已有一段时间。相比之下，核血红素生物合成很难描述。最近，已经提出了革兰氏阳性细菌的血红素生物合成途径，该途径填补了所有剩余的间隙。这是一组生物体，其中包括许多重要的病原体，这些病原体威胁公共卫生和生物形式，例如MRSA，结核病，炭疽和瘟疫的灾难性药物。该路径在最后三个步骤中与规范上的道路有所不同，其终点站最大的出发事件。最后一步是一种被称为Hemqs的酶催化的双氧化脱羧化：氯酸盐歧化酶的新型亚型（CLDS）。后者是血红素酶，将高氯酸盐呼吸的氯酸一终产物排毒，将其转化为Cl- o2。这项研究的初始阶段导致了对高氯酸盐呼吸器和非呼吸病原体的O2生成CLD的结构，机制和生物学的严格描述。该提案利用通过CLD的工作组装的工具，见解和科学团队，旨在在个人级别提供HEMQ功能的描述 分子并延伸到细胞环境。作为初步工作，已经产生了金黄色葡萄球菌的HEMQ菌株，并证明是血红素型型和小菌落变体（SCV）：一种与细胞内持久性和抗生素耐药性相关的表型。在串联中，来自金黄色葡萄球菌的HEMQ酶已显示出氧化性脱羧酸盐的二苯二羧酸盐，这是Coproheme III的四个丙酸酯侧链中的两个，该反应严格取决于H2O2。目的1专注于金黄色葡萄球菌系统，目的1是了解HEMQ如何结合并激活Coproheme朝着氧化物脱羧，从而产生与其底物（Coproheme III），Intermediate（HarderoHeme）（HarderoHeme）和产品（Heme B）（Heme B）的复杂的Sahemq的结构和能量模型。 AIM 2是测试HEMQ反应的机制，其中Coproheme在过氧化中既是底物和辅因子。时间分辨和动力学同位素方法将用于检查一系列唤起流铁氧化中间体和分子内氢原子转移的假设。最后，AIM 3使用遗传，基于细胞的和生化方法在细胞和进化的背景下了解HEMQ的功能。我们期望完成的工作完成，以定义对有氧生活至关重要的途径的最终步骤，这对于强大的致病性生长至关重要，并且在临床上与持久性和抗生素耐药性的发展相关。